河北省乳源金黄色葡萄球菌耐药性检测及耐药基因的研究

发布时间：2018-09-13 10:32

【摘要】：本试验主要研究河北省石家庄、保定、廊坊、承德、邢台、张家口、唐山7个地区集约化奶牛场临床分离的奶牛乳房炎病原菌-金黄色葡萄球菌(Staphylococcus aureus S.A)的主要生物学特性、耐药性及耐药基因的相关性。通过细菌生化试验及PCR方法的鉴定,分离出109株乳源金黄色葡萄球菌,按照世界卫生组织WHO推荐的Kirby-Bauer菌液涂布法对109株乳源金黄色葡萄球菌进行了38种抗生素的药物敏感试验,结果显示：109株乳源金黄色葡萄球菌对各类抗生素均有不同程度的耐药,且均表现为多重耐药,较为敏感的抗生素是头孢菌素类和氨基糖苷类,其中以头孢拉定、头孢氨苄、庆大霉素、阿米卡星、头孢吡肟、新霉素最为敏感；对磺胺类药物、青霉素类、林可胺类、大环内酯类和四环素类抗生素耐药严重,其中以磺胺异恶唑、氨苄西林、甲氧胺嘧啶、复方新诺明、阿莫西林、强力霉素、红霉素、克拉霉素、阿奇霉素、林可霉素耐药性最为严重。药敏试验为河北省区域内奶牛乳房炎的治疗提供科学的用药依据和用药指南。此外,本试验利用PCR技术对109株乳源金黄色葡萄球菌进行了8类抗生素耐药基因的检测,结果如下：(1)磺胺类耐药基因sull、su12、sul3阳性率分别为45.87%、6.42%、1.83%,有55株细菌含有该类耐药基因,总检出率为50.46%,无同时携带三种耐药基因的菌株；(2)四环素类耐药基因tetA、tetB、tetC、tetM阳性率分别为41.28%、18.35%、43.12%、10.09%,88株细菌扩增出四环素类耐药基因,总分离率为80.73%,有1株携带4种耐药基因,21株细菌未检出；(3)氨基糖类耐药基因aac(6')/aph(2")总检出率为66.97%,另有36株细菌未携带该类耐药基因；(4)p-内酰胺类耐药基因mecA的阳性率为0%,而femA阳性率分100%,推导试验菌株对β-内酰胺类抗生素耐药机制主要是由femA基因介导的；(5)大环内酯-林可胺类耐药基因ermA和ermC的阳性率分别为98.17%、62.39%,所有菌株均携带此类耐药基因,总检出率为100%；(6)喹诺酮类耐药基因norA,检出率为59.26%,随机选取30株耐喹诺酮类抗生素菌株,对其进行grlA与gyrA基因QRDR序列的检测,结果发现,grlA基因编码的第80位氨基酸发生突变,gyrA基因编码的第84位氨基酸发生突变,且有16株发生了gyrA和grlA双突变；(7)氯霉素类耐药基因cmlA、catl、flor其对应的阳性率分别为0%、22.02%、33.03%,未带任何氯霉素类耐药基因的菌株有55株,占50.46%；(8)糖肽类耐药基因vanA、vanB阳性率分别为66.97%和63.30%,有51株细菌均携带有两种耐药基因,占46.79%,未携带任何耐药基因的菌株有13株,占11.93%。以上各类药物耐药基因的检测,揭示了河北省乳源金黄色葡萄球菌主要耐药性机制,为河北省乳源金黄色葡萄球菌耐药性流行病学的调查提供了参考数据。
[Abstract]:The main biological characteristics of (Staphylococcus aureus S.A, a pathogen isolated from dairy cattle farms in 7 regions of Hebei Province, such as Shijiazhuang, Baoding, Langfang, Chengde, Xingtai, Zhangjiakou and Tangshan, were studied. Relationship between drug resistance and resistance genes. 109 strains of Staphylococcus aureus were isolated by biochemistry test and PCR method. According to the Kirby-Bauer liquid coating method recommended by WHO, the susceptibility tests of 38 kinds of antibiotics to 109 strains of Staphylococcus aureus were carried out. The results showed that 10 9 strains of milk source Staphylococcus aureus had different degrees of resistance to various antibiotics, and all of them showed multidrug resistance. The more sensitive antibiotics were cephalosporins and aminoglycosides, among which cefradine and cefalexin were the most sensitive antibiotics. Gentamicin, amikacin, cefepime, neomycin are the most sensitive; they are highly resistant to sulfonamides, penicillin, linamine, macrolides and tetracyclines, among which sulfamethoxazole, ampicillin, Methoxymine, compound sulfamine, amoxicillin, doxycycline, erythromycin, clarithromycin, azithromycin, lincomycin the most serious drug resistance. The drug sensitivity test provides scientific basis and guidance for the treatment of dairy cow mastitis in Hebei province. In addition, PCR technique was used to detect 8 antibiotic resistant genes in 109 strains of S. aureus from milk source. The results were as follows: (1) the positive rate of sull,su12,sul3 was 45.87% and 6.42% 1.83%, respectively, and 55 strains of bacteria contained these kinds of antibiotic resistance genes. The total positive rate of tetracycline resistance gene tetA,tetB,tetC,tetM was 41.28 and 18.355.The tetracycline resistance gene was amplified from 88 strains of bacteria, and the total detection rate was 50.46, and none of them carried three resistant genes at the same time; (2) the positive rate of tetracycline resistance gene was 41.28 and 18.355.The tetracycline resistance gene was amplified from 88 strains of bacteria. The total isolation rate was 80.73.The total detection rate of aac (6') / aph (2 ") was 66.97, while that of 36 strains of bacteria was not. (4) the positive rate of plactam resistance gene mecA was 0, while the positive rate of femA was 100%. The mechanism of 尾 -lactam antibiotic resistance was mainly mediated by femA gene. (5) the positive rates of ermA and ermC were 98.17% and 62.39%, respectively. (6) the detection rate of quinolone-resistant gene norA, was 59.26. 30 quinolone-resistant antibiotic strains were randomly selected and sequenced by grlA and gyrA gene QRDR. The results showed that the 84th amino acid mutation at the 80th amino acid position encoded by the grlA gene was mutated, and 16 strains had double mutations of gyrA and grlA. (7) the positive rates of chloramphenicol resistance gene cmlA,catl,flor were 0.22.02% and 33.03, 55 strains (50.46%) with no chloramphenicol resistance gene, (8) the positive rate of glycopeptide resistance gene vanA,vanB was 66.97% and 63.30%, respectively. There were 13 strains (11.93%) that did not carry any resistant genes. The detection of drug resistance genes revealed the main mechanism of drug resistance of Staphylococcus aureus in Hebei Province and provided reference data for the epidemiological investigation of drug resistance of Staphylococcus aureus in Hebei Province.
【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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